The use of the chemostat to study the relationship between cell growth rate, viability, and the effect of interferon on L 1210 cells

Mouse leukemia L 1210 cells were cultivated in the chemostat at growth rates ranging from 0.1 day⁻¹ (population doubling time (T_d) 166.3 h) to 2.0 day⁻¹ (T_d 8.3 h). At growth rates of 1.0 day⁻¹ and above, the viability of the steady-state culture was greater than 99%. However, below 1.0 day⁻¹ there was a progressive decrease in the viability of the culture with decreasing growth rate until a minimum growth rate (0.1 day⁻¹) was reached below which steady-state cultures of L 1210 cells could not be established. Interferon treatment had no effect on the viability (>99%) of L 1210 cells cultivated at fast growth rates in the chemostat, whereas at slow growth rates (0.35 day⁻¹) interferon treatment markedly reduced the viability of the culture, even though the percentage increase in the doubling time of interferon-treated cultures was the same for cells cultivated at both fast and slow growth rates. Thus, although interferon is not directly cytotoxic, it can cause cell death by reducing the rate of cell multiplication below the minimum value compatible with viability.     

Production of a self-activating CBM-factor X fusion protein in a stable transformed Sf9 insect cell line using high cell density perfusion culture

Factor Xa is a serine protease, whose high selectivity can be used to cleave protein tags from recombinant proteins. A fusion protein comprised of a self-activating form of factor X linked to a cellulose-binding module, saCBMFX, was produced in a stable transformed Sf9 insect cell line. The activity of the insect cell produced saCBMFX was higher than the equivalent mammalian cell produced material. A 1.5 l batch fermentation reached a maximum cell concentration of 1.6 × 10⁷ cells ml⁻¹ and a final saCBMFX concentration of 4 mg l⁻¹. The production of saCBMFX by this cell line was also analyzed in a 1.5 l perfusion system using an ultrasonic filter as a cell-retention device for flow rates up to 3.5 l day⁻¹. The cell-retention efficiency of an air backflush mode of acoustic filter operation was greater than 95% and eliminated the need to pump the relatively shear sensitive insect cells. In the perfusion system over 4 × 10⁷ Sf9 cells ml⁻¹ were obtained with a viability greater than 80%. With a doubling of viable cell concentration from 1.5 to 3 × 10⁷ cells ml⁻¹ the saCBMFX production rate was doubled to 6 mg l⁻¹ day⁻¹. The saCBMFX volumetric productivity of the perfusion system was higher than the batch fermentations (0.6 mg l⁻¹ day⁻¹) by an order of magnitude.     

An analysis of the growth of Gluconobacter oxydans in chemostat cultures

Gluconobacter oxydans was grown successively in glucose and nitrogen-limited chemostat cultures. Construction of mass balances of organisms growing at increasing dilution rates in glucose-limited cultures, at pH 5.5, revealed a major shift from extensive glucose metabolism via the pentose phosphate pathway to the direct pathway of glucose oxidation yielding gluconic acid. Thus, whereas carbon dioxide production from glucose accounted for 49.4% of the carbon input at a dilution rate (D)=0.05 h^-1, it accounted for only 1.3% at D=0.26 h^-1. This decline in pentose phosphate pathway activity resulted in decreasing molar growth yields on glucose. At dilution rates of 0.05 h^-1 and 0.26 h^-1 molar growth yields of 19.5 g/mol and 3.2 g/mol, respectively, were obtained. Increase of the steady state glucose concentration in nitrogen-limited chemostat cultures maintained at a constant dilution rate also resulted in a decreased flow of carbon through the pentose phosphate pathway. Above a threshold value of 15–20 mM glucose in the culture, pentose phosphate pathway activity almost completely inhibited. In G. oxydans the coupling between energy generation and growth was very inefficient; yield values obtained at various dilution rates varied between 0.8–3.4 g/cells synthesized per 0.5 mol of oxygen consumed.     

Continuous cultivation of the yeast Candida utilis at different dilution rates on pineapple cannery effluent

Candida utilis was grown on a pineapple cannery effluent in a chemostat at dilution rates ranging between 0.05 and 0.65 h^–1 to establish optimal conditions for biomass production and chemical oxygen demand (COD) reduction. Sucrose, fructose and glucose were the main sugars in the effluent. Maximum value for cell yield coefficient and productivity were (0.686, g_x/g_s) and (2.96, g_x/l/h) at a dilution rate of 0.425 and 0.475 h^–1, respectively, while maximum COD reduction (98%) was attained at a dilution rate of 0.1 h^–1. The maintenance coefficient attained a value of (0.093, g_s/g_x/h). An increase in dilution rate produced a higher protein content of the biomass.     

Influence of growth rate on the accumulation of ergosterol in yeast-cells in a phosphate limited continuous culture

Summary The influence of the growth rate on the accumulation of ergosterol inSaccharomyces cerevisiae was studied with glucose and ethanol as substrates under P-limitation in chemostat experiments. In cultures with glucose as carbon source a decrease in ergosterol content with dilution rates up to 0.08 h^–1 was observed, whereas above this dilution rate an increase in ergosterol content occurred. Similar but less marked effects were attained with ethanol as carbon source. A maximum specific rate of ergosterol synthesis of about 2.4 mg per h and g dry cell mass was calculated for phosphorus limited cultures.     

Effect of the dilution rate on the biomass yield ofBacillus thuringiensis and determination of its rate coefficients under steady-state conditions

Depending on the biomass yield on glucose and the cell morphology ofBacillus thuringiensis, three different metabolic states were observed in continuous culture. At dilution rates between 0.18 h^–1 and 0.31 h^–1 vegetative cells, sporulating bacteria and spores coexisted, while glucose and amino acids were consumed. Only vegetative cells were observed at dilution rates between 0.42 h^–1 and 0.47 h^–1 and glucose was used as the main carbon and energy source. AtD = 0.50 h^–1 the biomass yield on glucose decreases sharply. To define better the specific growth rate range in which the microorganism uses mainly glucose, a dilution rate of 0.25–0.45 h^–1 was studied. The experimental data could be adjusted to a Monod model and the following rate coefficients and growth yields were determined: maximum specific growth rate 0.54 h^–1, saturation constant 0.56 mg glucose ml^–1, biomass growth yields 0.43 g cells (g glucose)^–1, and 0.76 g cells (g oxygen)^–1, and maintenance coefficients 0.065 g glucose (g cells)^–1 h^–1 and 0.039 g oxygen (g cells)^–1 h^–1.     

Influence of Environmental Factors and Medium Composition on Vibrio gazogenes Growth and Prodigiosin Production

Vibrio gazogenes ATCC 29988 growth and prodigiosin synthesis were studied in batch culture on complex and defined media and in chemostat cultures on defined medium. In batch culture on complex medium, a maximum growth rate of 0.75 h⁻¹ and a maximum prodigiosin concentration of 80 ng of prodigiosin · mg of cell protein⁻¹ were observed. In batch culture on defined medium, maximum growth rates were lower (maximum growth rate, 0.40 h⁻¹), and maximum prodigiosin concentrations were higher (1,500 ng · mg of protein⁻¹). In batch culture on either complex or defined medium, growth was characterized by a period of logarithmic growth followed by a period of linear growth; on either medium, prodigiosin biosynthesis was maximum during linear growth. In batch culture on defined medium, the initial concentration of glucose optimal for growth and pigment production was 3.0%; higher levels of glucose suppressed synthesis of the pigment. V. gazogenes had an absolute requirement for Na⁺; optimal growth occurred in the presence of 100 mM NaCl. Increases in the concentration of Na⁺ up to 600 mM resulted in further increases in the concentration of pigment in the broth. Prodigiosin was synthesized at a maximum level in the presence of inorganic phosphate concentrations suboptimal for growth. Concentrations of KH₂PO₄ above 0.4 mM caused decreased pigment synthesis, whereas maximum cell growth occurred at 1.0 mM. Optimal growth and pigment production occurred in the presence of 8 to 16 mg of ferric ion · liter⁻¹, with higher concentrations proving inhibitory to both growth and pigment production. Both growth and pigment production were found to decrease with increased concentrations of p-aminobenzoic acid. The highest specific concentration of prodigiosin (3,480 ng · mg protein⁻¹) was observed in chemostat cultures at a dilution rate of 0.057 h⁻¹. The specific rate of prodigiosin production at this dilution rate was approximately 80% greater than that observed in batch culture on defined medium. At dilution rates greater than 0.057 h⁻¹, the concentration of cells decreased with increasing dilution rate, resulting in a profile comparable to that expected for linear growth kinetics. No explanation could be found for the linear growth profiles obtained for both batch and chemostat cultures.     

Growth and Antithraquinone Biosynthesis by Galium mollugo L. Cells in Batch and Chemostat Culture

The kinetics of growth, nutrient uptake, and anthraquinone biosynthesisby suspension cultures of Galium mollugo L. cells were examinedin batch and continuous (chemostat) culture. In batch culture,although the initial growth rate was constant (minimum doublingtime = 35 h) characteristic changes in cell composition wereobserved during the growth cycle particularly cell dry weight(between 3.9 and 9.2 g/10⁹ cells), cell anthraquinone (22–80mg/10⁹ cells), and cell protein (0.7–1.6 g/10⁹ cells).Using a chemostat steady state growth was established at twodifferent specific growth rates with mean doubling times of40 h and 25 h. Phosphate was established as the growth-limitingnutrient in chemostat culture at a concentration of 11 µgP ml^–1. In steady state growth at a doubling time of 40h the cell composition remained constant although this was differentfrom any cells grown in batch culture. The cell anthraquinonelevel in steady state growth was between 7 and 30 times lowerthan in batch culture. This result raises the question of therelative importance of growth rate and the growth-limiting nutrientin determining accumulation of secondary products by culturedplant cells.     

The relationship between transport kinetics and glucose uptake by Saccharomyces cerevisiae in aerobic chemostat cultures

The steady-state residual glucose concentrations in aerobic chemostat cultures of Saccharomyces cerevisiae ATCC 4126, grown in a complex medium, increased sharply in the respiro-fermentative region, suggesting a large increase in the apparent k_s value. By contrast, strain CBS 8066 exhibited much lower steady-state residual glucose concentrations in this region. Glucose transport assays were conducted with these strains to determine the relationship between transport kinetics and sugar assimilation. With strain CBS 8066, a high-affinity glucose uptake system was evident up to a dilution rate of 0.41 h^–1, with a low-affinity uptake system and high residual glucose levels only evident at the higher dilution rates. With strain ATCC 4126, the high-affinity uptake system was present up to a dilution rate of about 0.38 h^–1, but a low-affinity uptake system was discerned already from a dilution rate of 0.27 h^–1, which coincided with the sharp increase in the residual glucose concentration. Neither of the above yeast strains had an absolute vitamin requirement for aerobic growth. Nevertheless, in the same medium supplemented with vitamins, no low-affinity uptake system was evident in cells of strain ATCC 4126 even at high dilution rates and the steady-state residual glucose concentration was much lower. The shift in the relative proportions of the high and low-affinity uptake systems of strain ATCC 4126, which might have been mediated by an inositol deficiency through its effect on the cell membrane, may offer an explanation for the unusually high steady-state residual glucose concentrations observed at dilution rates above 52% of the wash-out dilution rate.     

Coevolution of Bacteriophage PP01 and Escherichia coli O157:H7 in Continuous Culture

The interaction between Escherichia coli O157:H7 and its specific bacteriophage PP01 was investigated in chemostat continuous culture. Following the addition of bacteriophage PP01, E. coli O157:H7 cell lysis was observed by over 4 orders of magnitude at a dilution rate of 0.876 h⁻¹ and by 3 orders of magnitude at a lower dilution rate (0.327 h⁻¹). However, the appearance of a series of phage-resistant E. coli isolates, which showed a low efficiency of plating against bacteriophage PP01, led to an increase in the cell concentration in the culture. The colony shape, outer membrane protein expression, and lipopolysaccharide production of each escape mutant were compared. Cessation of major outer membrane protein OmpC production and alteration of lipopolysaccharide composition enabled E. coli O157:H7 to escape PP01 infection. One of the escape mutants of E. coli O157:H7 which formed a mucoid colony (Mu) on Luria-Bertani agar appeared 56 h postincubation at a dilution rate of 0.867 h⁻¹ and persisted until the end of the experiment (~200 h). Mu mutant cells could coexist with bacteriophage PP01 in batch culture. Concentrations of the Mu cells and bacteriophage PP01 increased together. The appearance of mutant phage, which showed a different host range among the O157:H7 escape mutants than wild-type PP01, was also detected in the chemostat culture. Thus, coevolution of phage and E. coli O157:H7 proceeded as a mutual arms race in chemostat continuous culture.     

Kinetics of biphasic growth of yeast in continuous and fed-batch cultures

The influence of dilution rate on the production of biomass, ethanol, and invertase in an aerobic culture of Saccharomyces carlsbergensis was studied in a glucose-limited chemostat culture. A kinetic model was developed to analyze the biphasic growth of yeast on both the glucose remaining and the ethanol produced in the culture. The model assumes a double effect where glucose regulates the flux of glucose catabolism (respiration and aerobic fermentation) and the ethanol utilization in yeast cells. The model could successfully demonstrate the experimental results of a chemostat culture featuring the monotonic decrease of biomass concentration with an increase of dilution rate higher than 0.2 hr^?1 as well as the maximum ethanol concentration at a particular dilution rate around 0.5 hr^?1. Some supplementary data were collected from an ethanol-limited aerobic chemostat culture and a glucose-limited anaerobic chemostat culture to use in the model calculation. Some parametric constants of cell growth, ethanol production, and invertase formation were determined in batch cultures under aerobic and anaerobic states as summarized in a table in comparison with the chemostat data. Using the constants, a prediction of the optimal control of a glucose fed-batch yeast culture was conducted in connection with an experiment for harvesting a high yield of yeast cells with high invertase activity.     

Utilization of the cyanobacteria Anabaena sp. ATCC 33047 in CO2 removal processes

In this paper the utilization of the cyanobacteria Anabaena sp. in carbon dioxide removal processes is evaluated. For this, continuous cultures of this strain were performed at different dilution rates; alternatives for the recovery of the organic matter produced being also studied. A maximum CO₂ fixation rate of 1.45 g CO₂ L⁻¹ day⁻¹ was measured experimentally, but it can be increased up to 3.0 g CO₂ L⁻¹ day⁻¹ outdoors. The CO₂ is mainly transformed into exopolysaccharides, biomass representing one third of the total organic matter produced. Organic matter can be recovered by sedimentation with efficiencies higher than 90%, the velocity of sedimentation being 2 · 10⁻⁴ s⁻¹. The major compounds were carbohydrates and proteins with productivities of 0.70 and 0.12 g L⁻¹ day⁻¹, respectively. The behaviour of the cultures of Anabaena sp. has been modelized, also the characteristics parameters requested to design separation units being reported. Finally, to valorizate the organic matter as biofertilizers and biofuels is proposed.     

A study of polynucleotide phosphorylase production by <Emphasis Type="Italic">Escherichia coli</Emphasis> in a hollow fibre reactor

A 30-l hollow fibre reactor with continuous fermentation for cell recycling of Escherichia coli AS 1.183 was used to remove the inhibitory effects on cell growth and extend the fast growth phase to increase the yield of polynucleotide phosphorylase (PNPase) in E. coli cells. When the dilution rate was 1.5 h⁻¹, the cell concentration of E. coli reached 235 g/l (wet wt, 70% moisture content), with PNPase activity above 90 u/g (wet wt). With the dilution rate is 1.0 h⁻¹, the fermentor volumetric productivity of PNPase in a hollow fiber reactor can reach 974 (u/h * l) compared to 20 (u/h * l) in a conventional batch culture.     

The effect of molybdate and tungstate ions on the metabolic rates and enzyme activities in methanol-grown Methylobacterium sp. RXM

Feed-switching experiments were carried out in steady-state methanol-excess chemostat Methylobacterium sp. RXM cultures at a fixed dilution rate, temperature and pH (0.10 h^–1, 30° C and 6.95, respectively). The removal of molybdate from the nutrient supply led to a metabolic energy deficiency reflected in the molar growth yield and biomass values. High carbon conversion efficiency was linked with high formate dehydrogenase (FDH) activity and observed only when either molybdate or tungstate was added to the feed medium. A constant coenzyme ratio NAD⁺/K-ferri-cyanide linked to FDH activity was found during the enzyme stimulation period following the feed-switching experiment with tungstate addition, which suggests that both activities belong to the same enzyme. Quantitative metabolic responses (carbon conversion efficiency, methanol and O₂ consumption rates, CO₂ production rate and respiratory quotient) were measured in between steady-states just after the shift in the nutrient supply composition. Correspondence to: F. M. Gírio     

Influence of dilution rate on the morphology and technological properties ofLactobacillus delbrueckii subsp.bulgaricus

Lactobacillus delbrueckii subsp.bulgaricus ATCC 11842 was grown in a chemostat at 45°C and pH 5.5 using glucose as the carbon source, with the aim of optimizing biomass production. Cells were grown in a complex medium under nitrogen. At dilution rates lower than 0.18h^–1, it was difficult to keep steady-state conditions and pleomorphic forms were observed. The addition of 30mM Ca²⁺ and Mn²⁺ reverted the cells to normal shape: 30mM Mg²⁺ had no effect. Increasing the dilution rate resulted in normal morphology without the addition of any cations. Under these conditions, a maximum productivity of 1.24g dry biomass 1^–1 h^–1 was obtained. The maximum growth yield, corrected for maintenance, was 30g biomass mol^–1 glucose and the maintenance energy was 0.26g glucose g^–1 biomass h^–1. Lactate was the main fermentation product at all glucose concentrations used in the fed medium. Cells grown at high dilution rates had normal technological properties (acid production and proteolysis) when tested in milk.     

Protease and lipase production by a strain ofSerratia marcescens (532 S)

Summary Production of both exolipase and exoprotease activities bySerratia marcescens 532 S isolated from an aerobic fixed-biomass reactor were strongly influenced by nutritional factors which acted as inducers or repressors. In batch culture, protease and lipase activities were produced after the exponential phase. NH₄Cl, amino acids and simple carbon sources caused repression of protease activity. At a concentration of 1.5 g L^–1, the individual addition of maltose, mannitol, acetate, fructose or glucose, repressed exoprotease production, with the greatest effect by glucose. An inverse relationship existed between exoprotease synthesis and increasing glucose concentrations. Lipids activated lipase production, the most significant increase occurred when Tween 80 was added in the medium. Thus, glucidolytic, proteolytic and lipolytic activities could be efficiently expressed in batch cultures only successively.At low dilution rate of chemostat cultures with a constant glucose input concentration of 2 g L^–1, glucidolytic, proteolytic and lipolytic activities were produced, but did not have the same regulation: atD values <0.08 h^–1, the level of protease activity dropped while that of lipase showed a corresponding increase. Above these values, increasingD led to a decrease of the two hydrolase activities, at the level of the specific activities as well as in the specific rate of biosynthesis of each enzyme. Similar results were obtained in chemostat culture with a constant specific growth rate of 0.04 h^–1 with increasing glucose input concentrations, i.e. protease and lipase activities decreased when the specific glucose uptake rates were enhanced.     

Isolation and characterization of microcystin producing Microcystis from a Central Indian water bloom

Microcystis aeruginosa Kütz, a well-known microcystin (hepatotoxin) producing cyanobacterium was the dominant bloom-forming organism in a mesotrophic lake at Nagpur in Central India, which was isolated and characterized for morphospecies and microcystin content. Compact spherical colonies, formation of daughter colonies, and clathration of older colonies leading to release of solitary cells, were characteristics of laboratory grown M. aeruginosa. Its growth, monitored as increase in optical density (OD) measured at 678 nm (the wavelength selected using dilution curve technique), exhibited a maximum specific growth rate (μ_max) of 0.34 day⁻¹ which, was attained on the 5th day of the experiment with a doubling time of 3.25 days. Though the morphological characters of the M. aeruginosa under field conditions were not retained under laboratory conditions, the microcystin content and type of variants did match with bloom samples. Reverse phase high performance liquid chromatography (RP-HPLC) analyses revealed that the laboratory grown isolate of Microcystis produced microcystin-RR (732 μg g⁻¹ dry weight biomass) and demethylated microcystin-RR (165 μg g⁻¹ dry weight biomass) variants, which are reported to be less toxic when compared to microcystin-LR. LC/ESI/MS further confirmed the presence of these two variants. Geographical distribution of microcystin variants and their prevailing concentrations need to be considered during formulation of guideline values for drinking and recreational waters.     

Production of thermostable xylanases in batch and continuous culture by Thermomonospora fusca KW 3

Summary The actinomycete Thermomonospora fusca KW 3 produced novel thermostable xylanases in batch and continuous cultures in media containing insoluble xylan. The production of xylanases could be induced with oat spelt or beech xylan. Very low activities were detected when the strain was grown on glucose or xylose. In continuous cultivations, mycelial wall growth could be prevented using a stirrer speed controller. Homogeneous mixing of the insoluble substrate was obtained by vibrating the flexible tubes. T. fusca KW 3 could be grown on insoluble xylan at growth rates as high as 0.23 h^–1, equivalent to a doubling time of 3 h. Xylanase activity decreased from maximum levels of 2.5 units (U) ml^–1 with increasing dilution rate and was nearly constant at a level of 0.5 U ml^–1 with dilution rates greater than 0.1 h^–1. Correspondence to: P. Röthlisberger     

Metabolic and kinetic studies of hybridomas in exponentially fed-batch cultures using T-flasks

Exponentially fed-batch cultures (EFBC) of a murine hybridoma in T-flasks were explored as a simple alternative experimental tool to chemostats for the study of metabolism, growth and monoclonal antibody (MAb) production kinetics. EFBC were operated in the variable volume mode using an exponentially increasing and predetermined stepwise feeding profile of fresh complete medium. The dynamic and steady-state behaviors of the EFBC coincided with those reported for chemostats at dilution rates below the maximum growth rate. In particular, steady-state for growth rate and concentration of viable cells, glucose, and lactate was attained at different dilution rates between 0.005 and 0.05 h^–1. For such a range, the glucose and lactate metabolic quotients and the steady-state glucose concentration increased, whereas total MAb, volumetric, and specific MAb production rates decreased 65-, 6-, and 3-fold, respectively, with increasing dilution rates. The lactate from glucose yield remained relatively constant for dilution rates up to 0.03 h^–1, where it started to decrease. In contrast, viability remained above 80% at high dilution rates but rapidly decreased at dilution rates below 0.02 h^–1. No true washout occurred during operation above the maximum growth, as concluded from the constant viable cell number. However, growth rate decreased to as low as 0.01 h^–1, suggesting the requirement of a minimum cell density, and concomitant autocrine growth factors, for growth. Chemostat operation drawbacks were avoided by EFBC in T-flasks. Namely, simple and stable operation was obtained at dilution rates ranging from very low to above the maximum growth rate. Furthermore, simultaneous operation of multiple experiments in reduced size was possible, minimizing start-up time, media and equipment costs.Abbreviations EFBC exponentially-fed batch culture - CSC continuous suspended culture - MAb monoclonal antibody - D dilution rate - q _i metabolic quotient or specific rate of consumption or production of i     

A flow cytometry analysis of batch and continuous culture transient diauxic growth in Hansenula polymorpha

A flow cytometry analysis and in vitro enzyme activity study is carried out on the methylotrophic yeast, Hansenula polymorpha, during both (a) batch growth and (b) continuous cultures subjected to single perturbations in either system dilution rate or influent carbon substrate composition. Flow cytometry of yeasts growing diauxically on a glucose: methanol mixture during exponential growth, exhibit DNA and RNA distributions indicative of the S-synthesis-phase of the cell cycle. Cells at the stationary growth stage exhibit DNA and RNA distributions that indicate one portion of the population in the G ₀/G₁ resting phase and another in the M-mitosis-phase.Yeast cells grown at a steady-state of D=0.2 h¹, then shifted to D=0.35 h^–1, at a constant influent substrate mixture, are also examined with both flow cytometry and in vitro enzyme assays. Distributions of DNA, RNA, and total protein at either steady state and during the shift between dilution rates did not resemble any observed in batch culture. Flow cytometry indicates significant changes in cell composition within 20 min of the imposed dilution rate shift. In vitro enzyme assays show a response time in decreasing methanol oxidase activity of 2.5–3 h upon a dilution rate shift-up, while hexokinase activity increases to its steady-state level in less than 3 h. Similar cell compositional changes are reported for shifts in influent substrate methanol: glucose ratio at a constant dilution rate of D=0.35 h ^–1. Results suggest that an unsteady-state regime, oscillating between conditions that promote maximum enzyme activity of either glucose- or methanol-metabolizing enzymes, may allow simultaneous enhanced time-averaged production of both sets of enzymes.